Microorganisms adapt to a broad variety of environmental conditions. This versatility is characterized by the reorganization of macromolecular structure, the induction and/or suppression of enzyme systems, and the redistribution among cellular metabolic pools.
The theory and practice of "nutrient limitation" effects in fermentation systems is elaborated in literature such as Biochemical Engineering, Second Edition, Academic Press, New York, 1973; Biochemical Engineering Fundamentals, McGraw-Hill, New York, 1977; Principles Of Microbe And Cell Cultivation, John Wiley and Sons, New York, 1975; Fermentation And Enzyme Technology, John Wiley and Sons, New York, 1979; and the like.
Biotechnology and Bioengineering, 18, 180 (1976) is directed to transient response of Enterobacter aerogenes under a dual nutrient limitation in a chemostat. Quantitative evidence is provided that cells can be grown under dual nutrient limitation. The pattern of response is consistent with the hypothesis, for example, that phosphate-limitation restricts nucleic acid synthesis and that nitrogen-limitation restricts protein synthesis.
In a continuous fermentation (or chemostat) mode of cultivating microorganisms, growth nutrient-limitation is necessary in order to achieve a "steady state", i.e., a constant level of cell concentration in a continuous flow reactor with a defined medium concentration.
As indicated in the literature, conventional nutrient-limitation is primarily a technique to achieve steady state continuous fermentation and to study various yield and maintenance factors of cell mass with respect to various nutrients for cell growth. For the production of conventional fermentation products, such as ethanol, citric acid, lactic acid, acetic acid, and the like (primary metabolites), or antibiotics, microbial toxins, and the like (secondary metabolites) in a continuous flow reactor, nutrient-limitation can also be used to achieve steady state product formation. However, in a chemostat this type of nutrient-limitation has little or no effect on the stability of cells, i.e., the maintenance of the production and productivity level of a specific metabolite.
With a Pseudomonas putida type of mutant strain, the cells can grow on a preferred growth carbon and energy source (glucose, succinate or acetate) and convert a non-growth carbon source (e.g., toluene) to a product (e.g., muconic acid). The mutant strain at least initially is unable to grow on toluene as a carbon source. However, in the presence of toluene and other nutrients over a prolonged period of time (1-2 days), cells within the population "revert"; i.e., exhibit a parent strain ability to grow on toluene. Initially only a few cells revert, perhaps only a single cell, and eventually the reverted cell(s) grow and become the dominant cell type because of the selective ability to grow on both the growth carbon and the "non-growth" carbon sources that are present. This reversion problem is unique for genetically manipulated mutant microorganisms in bioconversion systems.
In addition to achievement of a stable population of a mutant microorganism for the production of a specific metabolite in a bioconversion system, it is desirable to establish and maintain a high productivity level of the specific metabolite.
Accordingly, it is an object of this invention to provide an improved fermentation process in which a population of a mutant microorganism is stabilized by suppression of cell reversion, and the specific productivity of an accumulating quantity of specific metabolite is increased.
It is another object of this invention to provide an improved fermentation process for bioconversion of toluene to accumulating muconic acid with increased specific productivity in a steady state continuous mode.
Other objects and advantages of the present invention shall become apparent from the accompanying description and examples.